The present invention relates to a simultaneous assay for glucose and urea with a single reagent by monitoring concurrent reactions which produce changes in the electromagnetic radiation absorbance characteristics of the sample. In one aspect, the invention relates to the simultaneous measurement of glucose and urea in blood serum by monitoring two concurrent reactions at two or more different wavelengths.
In the field of diagnostics, various assays are designed to identify or quantify an analyte, such as glucose or urea, which may be present in a sample material. Unfortunately the assay is usually only specific to one analyte even though it may be desirable to diagnose more than one analyte for any given sample. This leads to multiple testing on the same sample which increases diagnosis cost and decreases efficiency. It is therefore desirable to develop diagnostic testing which can identify or quantify multiple analytes in an efficient manner.
For example, glucose and urea are two of the more common tests performed in the clinical chemistry laboratory. Analysis of glucose is typically done using either a hexokinase or glucose oxidase method (Tietz, N. W., Textbook of Clinical Chemistry, 1986, p. 785). In the hexokinase method glucose is converted to glucose- 6 -phosphate hexokinase and adenosine triphosphate. Glucose-6 -phosphate then reacts with glucose- 6 -phosphate dehydrogenase (G-6-PDH) to produce 6 -phosphogluconate, with the concomitant reduction of nicotinamide-adenine dinucleotide (NAD) producing an increase in absorbance at 340 nm. The glucose oxidase method involves oxidation of glucose to gluconic acid and hydrogen peroxide by glucose oxidase. The hydrogen peroxide then reacts with peroxidase and a chromogenic oxygen acceptor to produce a color change in the 400-550 nm range. A third method for identifying glucose uses glucose dehydrogenase (Tietz, N. W., Textbook of Clinical Chemistry, 1986, p. 790). Glucose dehydrogenase converts glucose to gluconolactone with the concomitant reduction of NAD.
Analysis of urea is typically done using the urease/glutamate dehydrogenase method (Tietz, N. W., Textbook of Clinical Chemistry, 1986, p. 1268). Urease converts urea to ammonia and carbon dioxide. The ammonia produced reacts with glutamate dehydrogenase and alpha ketoglutarate to produce glutamate, with the concomitant oxidation of NADH producing a decrease in absorbance at 340 nm.
The assays mentioned above are performed with separate reagents in separate cuvettes. This costs the clinical chemistry lab time and money. By combining the two tests into one test the lab would be able to realize an increase in productivity and also a cost savings.
Combining the two tests is not a straightforward task. Reagents must be selected that allow precise measurement of one analyte (i.e., glucose), without interfering in the measurement of the second analyte (i.e., urea). For example, the combination of the glucose hexokinase and urea urease methods is eliminated by the fact that both use the NAD/NADH reaction. The combination of the glucose oxidase and urea urease methods is eliminated by the fact that peroxidase in the glucose reaction would oxidize the NADH in the urea reaction.
One way of combining the two assays in a single reaction vessel is to do a sequential assay such as disclosed in U.S. Pat. No. 4,425,427 to Luderer. European Application No. 133064 to Cam discloses another sequential assay where reagent for a first component is added to the vessel and at some later time a concentration is determined for the first component. Then a second reagent, which either quenches the first reaction or is added after the first reaction is complete, is added to the vessel to trigger a reaction with the second component. At some later time the concentration of the second component is determined. These reactions can either be monitored at the same wavelength or at different wavelengths (either through the use of filter wheels or diode arrays).
U.S. Pat. No. 3,925,162 describes the simultaneous measurement of enzyme activity in body fluids. In this approach the substrate for each of the enzymes to be identified are added to a reaction medium with other reagents and changes in the absorbance or fluorescence of the resulting reaction system are measured. The present invention is an approach where a single reagent system is used to simultaneously identify or quantify at least two analytes by monitoring the electromagnetic signal of the reaction mixture.